Compositions for the targeted insertion of a nucleotide sequence of interest into the genome of a plant

ABSTRACT

Compositions for the targeted integration of nucleotide sequences into a plant are provided. Plants and plant cells having multiple transfer cassettes stacked at precise locations in the genome of a plant or plant cell are provided. In specific embodiments, the plant or plant cell comprises at least two transfer cassettes. The first transfer cassette comprises a first nucleotide sequence of interest flanked by a first and a second recombination site, wherein the first and the second recombination sites are non-identical. The second transfer cassette comprises a second nucleotide sequence of interest flanked by the second recombination site and a third recombination site, wherein the third recombination site is non-identical to the first and said second recombination site. The second recombination site is shared between the first and the second transfer cassette, such that the genome of the plant cell comprises in the following order the first recombination site, the first nucleotide sequence of interest, the second recombination site, the second nucleotide sequence of interest, and the third recombination site.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/455,050, filed Dec. 6, 1999, which is a divisional application ofU.S. application Ser. No. 09/193,502 filed Nov. 17, 1998, now U.S. Pat.No. 6,187,994, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/065,627, filed Nov. 18, 1997, and U.S. Application Ser. No.60/065,613, filed Nov. 18, 1997, all of which are herein incorporated byreference.

FIELD OF THE INVENTION

The invention relates to the genetic modification of plants.Particularly, the control of gene integration and expression in plantsis provided.

BACKGROUND OF THE INVENTION

Genetic modification techniques enable one to insert exogenousnucleotide sequences into an organism's genome. A number of methods havebeen described for the genetic modification of plants. All of thesemethods are based on introducing a foreign DNA into the plant cell,isolation of those cells containing the foreign DNA integrated into thegenome, followed by subsequent regeneration of a whole plant.Unfortunately, such methods produce transformed cells that contain theintroduced foreign DNA inserted randomly throughout the genome and oftenin multiple copies.

The random insertion of introduced DNA into the genome of host cells canbe lethal if the foreign DNA happens to insert into, and thus mutate, acritically important native gene. In addition, even if a randominsertion event does not impair the functioning of a host cell gene, theexpression of an inserted foreign gene may be influenced by “positioneffects” caused by the surrounding genomic DNA. In some cases, the geneis inserted into sites where the position effects are strong enough toprevent the synthesis of an effective amount of product from theintroduced gene. In other instances, overproduction of the gene producthas deleterious effects on the cell.

Transgene expression is typically governed by the sequences, includingpromoters and enhancers, which are physically linked to the transgene.Currently, it is not possible to precisely modify the structure oftransgenes once they have been introduced into plant cells. In manyapplications of transgene technology, it would be desirable to introducethe transgene in one form, and then be able to modify the transgene in adefined manner. By this means, transgenes could be activated orinactivated where the sequences that control transgene expression can bealtered by either removing sequences present in the original transgeneor by inserting additional sequences into the transgene.

For higher eukaryotes, homologous recombination is an essential eventparticipating in processes like DNA repair and chromatid exchange duringmitosis and meiosis. Recombination depends on two highly homologousextended sequences and several auxiliary proteins. Strand separation canoccur at any point between the regions of homology, although particularsequences may influence efficiency. These processes can be exploited fora targeted integration of transgenes into the genome of certain celltypes.

Even with the advances in genetic modification of higher plants, themajor problems associated with the conventional gene transformationtechniques have remained essentially unresolved as to the problemsdiscussed above relating to variable expression levels due tochromosomal position effects and copy number variation of transferredgenes. For these reasons, efficient methods are needed for targeting andcontrol of insertion of nucleotide sequences to be integrated into aplant genome.

SUMMARY OF THE INVENTION

Compositions and methods for the targeted integration of nucleotidesequences into a transformed plant are provided. The compositionscomprise transfer cassettes which are flanked by non-identicalrecombination sites.

The methods find use in targeting the integration of nucleotidesequences of interest to a specific chromosomal site, finding optimalintegration sites in a plant genome, comparing promoter activity intransformed plants, engineering chromosomal rearrangements, and othergenetic manipulation of plants.

Novel minimal recombination sites (FRT) are provided for use in themethods of the invention. Also provided are targeting cassettes andtransgenic plants and plant cells containing corresponding non-identicalrecombination sites.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides one scheme for gene stacking via site-specificintegration using the FLP system.

FIG. 2 provides a construct of the representative plasmid PHP10616.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for the directional, targeted integration ofexogenous nucleotides into a transformed plant are provided. The methodsuse novel recombination sites in a gene targeting system whichfacilitates directional targeting of desired genes and nucleotidesequences into corresponding recombination sites previously introducedinto the target plant genome.

In the methods of the invention, a nucleotide sequence flanked by twonon-identical recombination sites is introduced into the targetorganism's genome establishing a target site for insertion of nucleotidesequences of interest. Once a stable plant or cultured tissue isestablished a second construct, or nucleotide sequence of interest,flanked by corresponding recombination sites as those flanking thetarget site, is introduced into the stably transformed plant or tissuesin the presence of a recombinase protein. This process results inexchange of the nucleotide sequences between the non-identicalrecombination sites of the target site and the transfer cassette.

It is recognized that the transformed plant may comprise multiple targetsites; i.e., sets of non-identical recombination sites. In this manner,multiple manipulations of the target site in the transformed plant areavailable. By target site in the transformed plant is intended a DNAsequence that has been inserted into the transformed plant's genome andcomprises non-identical recombination sites.

Examples of recombination sites for use in the invention are known inthe art and include FRT sites (See, for example, Schlake and Bode (1994)Biochemistry 33:12746-12751; Huang et al. (1991) Nucleic Acids Research19:443-448; Sadowski, Paul D. (1995) In Progress in Nucleic AcidResearch and Molecular Biology vol. 51, pp. 53-91; Michael M. Cox (1989)In Mobile DNA, Berg and Howe (eds) American Society of Microbiology,Washington D.C., pp. 116-670; Dixon et al. (1995) 18:449-458; Umlauf andCox (1988) The EMBO Journal 7:1845-1852; Buchholz et al. (1996) NucleicAcids Research 24:3118-3119; Kilby et al. (1993) Trends Genet.9:413-421: Rossant and Geagy (1995) Nat. Med. 1: 592-594; Albert et al.(1995) The Plant J. 7:649-659: Bayley et al. (1992) Plant Mol. Biol.18:353-361; Odell et al. (1990) Mol. Gen. Genet. 223:369-378; and Daleand Ow (1991) Proc. Natl. Acad. Sci. USA 88:10558-105620; all of whichare herein incorporated by reference.); Lox (Albert et al. (1995) PlantJ. 7:649-659; Qui et al. (1994) Proc. Natl. Acad. Sci. USA 91:1706-1710;Stuurman et al. (1996) Plant Mol. Biol. 32:901-913; Odell et al. (1990)Mol. Gen. Gevet. 223:369-378; Dale et al. (1990) Gene 91:79-85; andBayley et al. (1992) Plant Mol. Biol. 18:353-361.)

The two-micron plasmid found in most naturally occurring strains ofSaccharomyces cerevisiae, encodes a site-specific recombinase thatpromotes an inversion of the DNA between two inverted repeats. Thisinversion plays a central role in plasmid copy-number amplification. Theprotein, designated FLP protein, catalyzes site-specific recombinationevents. The minimal recombination site (FRT, SEQ ID NO:1) has beendefined and contains two inverted 13-base pair (bp) repeats surroundingan asymmetric 8-bp spacer. The FLP protein cleaves the site at thejunctions of the repeats and the spacer and is covalently linked to theDNA via a 3′ phosphate.

Site specific recombinases like FLP cleave and religate DNA at specifictarget sequences, resulting in a precisely defined recombination betweentwo identical sites. To function, the system needs the recombinationsites and the recombinase. No auxiliary factors are needed. Thus, theentire system can be inserted into and function in plant cells.

The yeast FLP\FRT site specific recombination system has been shown tofunction in plants. To date, the system has been utilized for excisionof unwanted DNA. See, Lyznik et al. (1993) Nucleic Acid Res. 21:969-975.In contrast, the present invention utilizes non-identical FRTs for theexchange, targeting, arrangement, insertion and control of expression ofnucleotide sequences in the plant genome.

To practice the methods of the invention, a transformed organism ofinterest, particularly a plant, containing a target site integrated intoits genome is needed. The target site is characterized by being flankedby non-identical recombination sites. A targeting cassette isadditionally required containing a nucleotide sequence flanked bycorresponding non-identical recombination sites as those sites containedin the target site of the transformed organism. A recombinase whichrecognizes the non-identical recombination sites and catalyzessite-specific recombination is required.

It is recognized that the recombinase can be provided by any means knownin the art. That is, it can be provided in the organism or plant cell bytransforming the organism with an expression cassette capable ofexpressing the recombinase in the organism, by transient expression; orby providing messenger RNA (mRNA) for the recombinase or the recombinaseprotein.

By “non-identical recombination sites” is intended that the flankingrecombination sites are not identical in sequence and will not recombineor recombination between the sites will be minimal. That is, oneflanking recombination site may be a FRT site where the secondrecombination site may be a mutated FRT site. The non-identicalrecombination sites used in the methods of the invention prevent orgreatly suppress recombination between the two flanking recombinationsites and excision of the nucleotide sequence contained therein.Accordingly, it is recognized that any suitable non-identicalrecombination sites may be utilized in the invention, including FRT andmutant FRT sites, FRT and LOX sites, LOX and mutant LOX sites, as wellas other recombination sites known in the art.

By suitable non-identical recombination site implies that in thepresence of active recombinase, excision of sequences between twonon-identical recombination sites occurs, if at all, with an efficiencyconsiderably lower than the recombinationally-mediated exchangetargeting arrangement of nucleotide sequences into the plant genome.Thus, suitable non-identical sites for use in the invention includethose sites where the efficiency of recombination between the sites islow; for example, where the efficiency is less than about 30 to about50%, preferably less than about 10 to about 30%, more preferably lessthan about 5 to about 10%.

As noted above, the recombination sites in the targeting cassettecorrespond to those in the target site of the transformed plant. Thatis, if the target site of the transformed plant contains flankingnon-identical recombination sites of FRT and a mutant FRT, the targetingcassette will contain the same FRT and mutant FRT non-identicalrecombination sites.

It is furthermore recognized that the recombinase, which is used in theinvention, will depend upon the recombination sites in the target siteof the transformed plant and the targeting cassette. That is, if FRTsites are utilized, the FLP recombinase will be needed. In the samemanner, where lox sites are utilized, the Cre recombinase is required.If the non-identical recombination sites comprise both a FRT and a loxsite, both the FLP and Cre recombinase will be required in the plantcell.

The FLP recombinase is a protein that catalyzes a site-specific reactionthat is involved in amplifying the copy number of the two micron plasmidof S. cerevisiae during DNA replication. FLP protein has been cloned andexpressed. See, for example, Cox (1993) Proc. Natl. Acad. Sci. U.S.A.80:4223-4227. The FLP recombinase for use in the invention may be thatderived from the genus Saccharomyces. It may be preferable to synthesizethe recombinase using plant preferred codons for optimum expression in aplant of interest. See, for example, U.S. application Ser. No.08/972,258 filed Nov. 18, 1997, entitled “Novel Nucleic Acid SequenceEncoding FLP Recombinase” now U.S. Pat. No. 5,929,301, hereinincorporated by reference.

The bacteriophage recombinase Cre catalyzes site-specific recombinationbetween two lox sites. The Cre recombinase is known in the art. See, forexample, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J.Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet.22:477-488; and Shaikh et al. (1977) J. Biol. Chem. 272:5695-5702. Allof which are herein incorporated by reference. Such Cre sequence mayalso be synthesized using plant preferred codons.

Where appropriate, the nucleotide sequences to be inserted in the plantgenome may be optimized for increased expression in the transformedplant. Where mammalian, yeast, or bacterial genes are used in theinvention, they can be synthesized using plant preferred codons forimproved expression. It is recognized that for expression in monocots,dicot genes can also be synthesized using monocot preferred codons.Methods are available in the art for synthesizing plant preferred genes.See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

The plant preferred codons may be determined from the codons utilizedmore frequently in the proteins expressed in the plant of interest. Itis recognized that monocot or dicot preferred sequences may beconstructed as well as plant preferred sequences for particular plantspecies. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlaket al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328; and Murray etal. (1989) Nucleic Acids Research, 17: 477-498. U.S. Pat. No. 5,380,831;U.S. Pat. No. 5,436,391; and the like, herein incorporated by reference.It is further recognized that all or any part of the gene sequence maybe optimized or synthetic. That is, fully optimized or partiallyoptimized sequences may also be used.

Additional sequence modifications are known to enhance gene expressionin a cellular host and can be used in the invention. These includeelimination of sequences encoding spurious polyadenylation signals,exon-intron splice site signals, transposon-like repeats, and other suchwell-characterized sequences, which may be deleterious to geneexpression. The G-C content of the sequence may be adjusted to levelsaverage for a given cellular host, as calculated by reference to knowngenes expressed in the host cell. When possible, the sequence ismodified to avoid predicted hairpin secondary mRNA structures.

The present invention also encompasses novel FLP recombination targetsites (FRT). The FRT (SEQ ID NO:1) has been identified as a minimalsequence comprising two 13 base pair repeats, separated by an 8 basespacer, as follows:

-   -   5′-GAAGTTCCTATTC[TCTAGAAA]GTATAGGAACTTC3′        wherein the nucleotides within the brackets indicate the spacer        region. The nucleotides in the spacer region can be replaced        with a combination of nucleotides, so long as the two 13-base        repeats are separated by eight nucleotides. It appears that the        actual nucleotide sequence of the spacer is not critical,        however for the practice of the invention, some substitutions of        nucleotides in the space region may work better than others.

The eight base pair spacer is involved in DNA-DNA pairing during strandexchange. The asymmetry of the region determines the direction of sitealignment in the recombination event, which will subsequently lead toeither inversion or excision. As indicated above, most of the spacer canbe mutated without a loss of function. See, for example, Schlake andBode (1994) Biochemistry 33:12746-12751, herein incorporated byreference.

Novel FRT mutant sites are provided for use in the practice of themethods of the present invention. Such mutant sites may be constructedby PCR-based mutagenesis. While mutant FRT sites (SEQ ID NOS:2, 3, 4 and5) are provided herein, it is recognized that other mutant FRT sites maybe used in the practice of the invention. The present invention is notthe use of a particular FRT or recombination site, but rather thatnon-identical recombination sites or FRT sites can be utilized fortargeted insertion and expression of nucleotide sequences in a plantgenome. Thus, other mutant FRT sites can be constructed and utilizedbased upon the present disclosure.

As discussed above, bringing genomic DNA containing a target site withnon-identical recombination sites together with a vector containing atransfer cassette with corresponding non-identical recombination sites,in the presence of the recombinase, results in recombination. Thenucleotide sequence of the transfer cassette located between theflanking recombination sites is exchanged with the nucleotide sequenceof the target site located between the flanking recombination sites. Inthis manner, nucleotide sequences of interest may be preciselyincorporated into the genome of the host.

It is recognized that many variations of the invention can be practiced.For example, target sites can be constructed having multiplenon-identical recombination sites. Thus, multiple genes or nucleotidesequences can be stacked or ordered at precise locations in the plantgenome. Likewise, once a target site has been established within thegenome, additional recombination sites may be introduced byincorporating such sites within the nucleotide sequence of the transfercassette and the transfer of the sites to the target sequence. Thus,once a target site has been established, it is possible to subsequentlyadd sites, or alter sites through recombination.

Another variation includes providing a promoter or transcriptioninitiation region operably linked with the target site in an organism.Preferably, the promoter will be 5′ to the first recombination site. Bytransforming the organism with a transfer cassette comprising a codingregion, expression of the coding region will occur upon integration ofthe transfer cassette into the target site. This embodiment provides fora method to select transformed cells, particularly plant cells, byproviding a selectable marker sequence as the coding sequence.

Other advantages of the present system include the ability to reduce thecomplexity of integration of trans-genes or transferred DNA in anorganism by utilizing transfer cassettes as discussed above andselecting organisms with simple integration patterns. In the samemanner, preferred sites within the genome can be identified by comparingseveral transformation events. A preferred site within the genomeincludes one that does not disrupt expression of essential sequences andprovides for adequate expression of the transgene sequence.

The methods of the invention also provide for means to combine multiplecassettes at one location within the genome. See, for example, FIG. 1.Recombination sites may be added or deleted at target sites within thegenome.

Any means known in the art for bringing the three components of thesystem together may be used in the invention. For example, a plant canbe stably transformed to harbor the target site in its genome. Therecombinase may be transiently expressed or provided. Alternatively, anucleotide sequence capable of expressing the recombinase may be stablyintegrated into the genome of the plant. In the presence of thecorresponding target site and the recombinase, the transfer cassette,flanked by corresponding non-identical recombination sites, is insertedinto the transformed plant's genome.

Alternatively, the components of the system may be brought together bysexually crossing transformed plants. In this embodiment, a transformedplant, parent one, containing a target site integrated in its genome canbe sexually crossed with a second plant, parent two, that has beengenetically transformed with a transfer cassette containing flankingnon-identical recombination sites, which correspond to those in plantone. Either plant one or plant two contains within its genome anucleotide sequence expressing recombinase. The recombinase may be underthe control of a constitutive or inducible promoter.

Inducible promoters include heat-inducible promoters,estradiol-responsive promoters, chemical inducible promoters, and thelike. Pathogen inducible promoters include those frompathogenesis-related proteins (PR proteins), which are induced followinginfection by a pathogen; e.g., PR proteins, SAR proteins,beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al.(1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) The PlantCell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. In thismanner, expression of recombinase and subsequent activity at therecombination sites can be controlled.

Constitutive promoters for use in expression of genes in plants areknown in the art. Such promoters include, but are not limited to 35Spromoter of cauliflower mosaic virus (Depicker et al. (1982) Mol. Appl.Genet. 1:561-573; Odell et al. (1985) Nature 313:810-812), ubiquitinpromoter (Christensen et al. (1992) Plant Mol. Biol. 18:675-689),promoters from genes such as ribulose bisphosphate carboxylase (DeAlmeida et al. (1989) Mol. Gen. Genet. 218:78-98), actin (McElroy et al.(1990) Plant J. 2:163-171), histone, DnaJ (Baszczynski et al. (1997)Maydica 42:189-201), and the like.

The compositions and methods of the invention find use in targeting theintegration of transferred nucleotide sequences to a specificchromosomal site. The nucleotide sequence may encode any nucleotidesequence of interest. Particular genes of interest include those whichprovide a readily analyzable functional feature to the host cell and/ororganism, such as marker genes, as well as other genes that alter thephenotype of the recipient cells, and the like. Thus, genes effectingplant growth, height, susceptibility to disease, insects, nutritionalvalue, and the like may be utilized in the invention. The nucleotidesequence also may encode an “antisense” sequence to turn off or modifygene expression.

It is recognized that the nucleotide sequences will be utilized in afunctional expression unit or cassette. By functional expression unit orcassette is intended, the nucleotide sequence of interest with afunctional promoter, and in most instances a termination region. Thereare various ways to achieve the functional expression unit within thepractice of the invention. In one embodiment of the invention, thenucleic acid of interest is transferred or inserted into the genome as afunctional expression unit. Alternatively, the nucleotide sequence maybe inserted into a site within the genome which is 3′ to a promoterregion. In this latter instance, the insertion of the coding sequence 3′to the promoter region is such that a functional expression unit isachieved upon integration.

For convenience, for expression in plants, the nucleic acid encodingtarget sites and the transfer cassettes, including the nucleotidesequences of interest, can be contained within expression cassettes. Theexpression cassette will comprise a transcriptional initiation region,or promoter, operably linked to the nucleic acid encoding the peptide ofinterest. Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the gene or genes of interest to beunder the transcriptional regulation of the regulatory regions.

The transcriptional initiation region, the promoter, may be native orhomologous or foreign or heterologous to the host, or could be thenatural sequence or a synthetic sequence. By foreign is intended thatthe transcriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced. Either anative or heterologous promoter may be used with respect to the codingsequence of interest.

The transcriptional cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the potato proteinaseinhibitor (PinII) gene or from Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See also,Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987)Nucleic Acid Res. 15:9627-9639.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picomavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al. (1989)PNAS USA, 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology, 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak and Sarnow (1991) Nature, 353:90-94;untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4), (Jobling and Gehrke (1987) Nature, 325:622-625; tobaccomosaic virus leader (TMV), (Gallie et al. (1989) Molecular Biology ofRNA, pages 237-256, Gallie et al. (1987) Nucl. Acids Res. 15:3257-3273;and maize chlorotic mottle virus leader (MCMV) (Lommel, S. A. et al.(1991) Virology, 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiology, 84:965-968. Other methods known to enhance translation canalso be utilized, for example, introns, and the like.

The expression cassettes may contain one or more than one gene ornucleic acid sequence to be transferred and expressed in the transformedplant. Thus, each nucleic acid sequence will be operably linked to 5′and 3′ regulatory sequences. Alternatively, multiple expressioncassettes may be provided.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues.

See generally, Yarranton, G. T. (1992) Curr. Opin. Biotech., 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA, 89:6314-6318;Yao et al. (1992) Cell, 71:63-72; Reznikoff, W. S. (1992) Mol.Microbiol., 6:2419-2422; Barkley et al. (1980) The Operon, pp. 177-220;Hu et al. (1987) Cell, 48:555-566; Brown et al. (1987) Cell, 49:603-612;Figge et al. (1988) Cell, 52:713-722; Deuschle et al. (1989) Proc. Natl.Acad. Aci. USA, 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad.Sci. USA, 86:2549-2553; Deuschle et al. (1990) Science, 248:480-483;Gossen, M. (1993) PhD Thesis, University of Heidelberg; Reines et al.(1993) Proc. Natl. Acad. Sci. USA, 90:1917-1921; Labow et al. (1990)Mol. Cell Bio., 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad.Sci. USA, 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA,88:5072-5076; Wyborski et al. (1991) Nuc. Acids Res., 19:4647-4653;Hillenand-Wissman, A. (1989) Topics in Mol. and Struc. Biol.,10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.,35:1591-1595; Kleinschnidt et al. (1988) Biochemistry, 27:1094-1104;Gatz et al. (1992) Plant J., 2:397-404; Bonin, A. L. (1993) PhD Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA, 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.,36:913-919; Hlavka et al. (1985) Handbook of Exp. Pharmacology, 78; Gillet al. (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The methods of the invention can also be utilized to find optimalintegration sites within a plant genome. In this manner, a plant istransformed with an expression cassette comprising a selectable markergene. The expression cassette is a target site as the marker gene isflanked by non-identical recombination sites. Transformed protoplast,tissues, or whole plants can be tested to determine the levels ofactivity of the inserted gene. By comparison of cellular activities ofthe gene in different insertion sites, preferred integration sites maybe found wherein the gene is expressed at high or acceptable levels.These plants can then be utilized with subsequent retargeting techniquesto replace the marker gene with other genes or nucleotide sequences ofinterest. In the same manner, multiple genes may be inserted at theoptimal site for expression. See, for example, FIG. 2 which sets forthone scheme for gene stacking utilizing site-specific integration usingthe FRT/FLP system.

A variety of genetic manipulations are available using the compositionsof the present invention including, for example, comparing promoteractivity in a transformed plant. Prior to the present invention,promoter activity could not accurately be assessed and compared becausethe chimeric genes were inserted at different locations within the plantgenome. Such chromosomal locations affected activity. By utilizing themethods of the present invention, a direct comparison of promotoractivity in a defined chromosomal context is possible. Thus, using themethods, enhanced activity of genes can be achieved by selecting optimalchromosomal sites as well as optimal promoters for expression in theplant cell.

The present invention may be used for transformation of any plantspecies, including but not limited to corn (Zea mays), canola (Brassicanapus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers which may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Preferably, plants of the present invention are crop plants (forexample, corn, alfalfa, sunflower, canola, soybean, cotton, peanut,sorghum, wheat, tobacco, etc.), more preferably corn and soybean plants,yet more preferably corn plants.

It is recognized that the methods of the invention may be applied in anyplant system. Methods for transformation of plants are known in the art.In this manner, genetically modified plants, plant cells, plant tissue,seed, and the like can be obtained. Transformation protocols may varydepending on the type of plant or plant cell, i.e., monocot or dicot,targeted for transformation. Suitable methods of transforming plantcells include microinjection (Crossway et al. (1986) Biotechniques4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.USA, 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al.(1988) Biotechnology, 6:915-921), direct gene transfer (Paszkowski etal. (1984) EMBO J., 3:2717-2722), and ballistic particle acceleration(see, for example, Sanford et al., U.S. Pat. No. 4,945,050; WO91/10725and McCabe et al. (1988) Biotechnology, 6:923-926). Also see, Weissingeret al. (1988) Annual Rev. Genet., 22:421-477; Sanford et al. (1987)Particulate Science and Technology, 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology, 6:923-926 (soybean); Datta et al. (1990) Biotechnology,8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA,85:4305-4309 (maize); Klein et al. (1988) Biotechnology, 6:559-563(maize); WO91/10725 (maize); Klein et al. (1988) Plant Physiol.,91:440-444 (maize); Fromm et al. (1990) Biotechnology, 8:833-839; andGordon-Kamm et al. (1990) Plant Cell, 2:603-618 (maize); Hooydaas-VanSlogteren & Hooykaas (1984) Nature (London), 311:763-764; Bytebier etal. (1987) Proc. Natl. Acad. Sci. USA, 84:5345-5349 (Liliaceae); De Wetet al. (1985) In The Experimental Manipulation of Ovule Tissues, ed. G.P. Chapman et al., pp. 197-209. Longman, N.Y. (pollen); Kaeppler et al.(1990) Plant Cell Reports, 9:415-418; and Kaeppler et al. (1992) Theor.Appl. Genet., 84:560-566 (whisker-mediated transformation); D'Halluin etal. (1992) Plant Cell, 4:1495-1505 (electroporation); Li et al. (1993)Plant Cell Reports, 12:250-255 and Christou and Ford (1995) Annals ofBotany, 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology,14:745-750 (maize via Agrobacterium tumefaciens); all of which areherein incorporated by reference.

The cells which have been transformed may be grown into plants inaccordance with conventional approaches. See, for example, McCormick etal. (1986) Plant Cell Reports, 5:81-84. These regenerated plants maythen be pollinated with either the same transformed strain or differentstrains, and the resulting hybrid having the desired phenotypiccharacteristic identified. Two or more generations may be grown toensure that the subject phenotypic characteristic is stably maintainedand inherited and then seeds harvested to ensure the desired phenotypeor other property has been achieved.

It is recognized that any means of transformation may be utilized forthe present invention. However, for inserting the target site within thetransformed plant, Agrobacterium-mediated transformation may bepreferred. Agrobacterium-mediated transformation generally tends toinsert a lower copy number of transferred DNA than does particlebombardment or other transformation means.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The general present invention provides a procedure for using existingand novel FRT sites in a new gene targeting system which facilitatesdirectional retargeting of desired genes into FRT sites previouslyintroduced in the target organism's genome. The novel FRT sites differfrom previously described FRT sites in the sequence of the 8 bp spacerregions of the FRT sites. Previous publications also have shown that inthe presence of FLP protein, recombination of sequences between two FRTsites occurs efficiently only with two identical FRT sites. See forexample Umlauf and Cox (1988) Embo J. 7:1845-1852; Schlake and Bode(1994) Biochem. 33:12746-12751. To use the invention, a gene or DNAsequence is flanked by two non-identical FRT sites and introduced into atarget organism's genome. The enclosed gene can be a selectable marker,thereby allowing selection for successfully introduced sequences.Molecular characterization confirms integration of desired sequencesincluding complete FRT sites. Listed below are generic examples ofvector constructions useful in practicing the invention:

-   A. FRTa-P1-G1-T1-FRTb-   B. FRTa-P1-G1-T1-FRTa-   C. FRTb-P1-G1-T1-FRTb-   D. P1-FRTa-G1-T1-FRTb-   E. P1-FRTa-G1-T1-FRTa-   F. P1-FRTb-G1-T1-FRTb-   G. P1-ATG::FRTa::G1(noATG)-T1-P2-G2-T2-FRTb-   H. P1-ATG::FRTa::G1(noATG)-T1-P2-G2-T2-FRTb-P3-G3-T3-   I. P1-ATG::FRTa::G1(noATG)-T1-FRTa::G2(noATG)-T2-FRTb-   J. P1-ATG::FRTa::G1(noATG)-T1-FRTa::G2(noATG)-T2-FRTb-P3-G3-T3-   K. P1-FRTa-G1-T1-P2-G2-T2-FRTb-   L. P1-FRTa-G1-T1-P2-G2-T2-FRTb-P3-G3-T3-   M. P1-FRTa-G1-T1-FRTa-G2-T2-FRTb-   N. P1-FRTa-G1-T1-FRTa-G2-T2-FRTb-P3-G3-T3    Variations thereof may be constructed with other promoters, genes,    terminators or FRT sites.

FRTa and FRTb are two examples of non-identical FRT sites. P1, P2 and P3are different promoters, G1, G2, and G3 are different genes, T1, T2 andT3 are different terminators. ATG is the start of translation codon forthe subsequent gene. The designation noATG indicates that particulargene is devoid of the ATG translation start codon. The symbol :: impliesa fusion between adjacent elements, and where used between ATG, FRT anda gene, implies that the sequences are put together to generate an inframe translation fusion that results in a properly expressed andfunctional gene product.

A to F are preferred configurations for testing new FRT sites forability to recombine sequences between them; the desired situation beingthat when two of the same site are used, recombination is efficient andthat when two different sites are used, no recombination between themtakes place in the presence of FLP protein. G to J are preferredconfigurations for general use in developing lines for retargeting. Itis understood that any number of genes or other combinations ofsequences can be assembled for use as part of this invention. K to N arepossible configurations that could be used also.

Once a stable plant or cultured tissue is established with one of theconstructs above, a second construct flanked by the same FRT sites usedto flank the sequences in the first construct above is introduced intothe stably transformed tissues in conjunction with the expression of FLPprotein. The new vector constructs can be, but are not limited to thefollowing:

-   O. FRTa::G1(noATG)-T1-FRTb-   P. FRTa::G1(noATG)-T1-P2-G2-T2-FRTb-   Q. FRTa-G1-T1-FRTb-   R. FRTa-G1-T1-P2-G2-T2-FRTb    The FLP protein can be supplied by a) co-transforming with a plasmid    carrying a gene encoding FLP; b) co-introducing FLP mRNA or protein    directly; c) using a line for the initial transformation that    expresses FLP either constitutively or following induction; or d)    growing out the plants carrying the initial targeted vectors,    crossing to plants that express active FLP protein and selecting    events in the progeny.

As a working example, sequence O above is introduced into a linecontaining a copy of sequence G stably integrated in the genome, in thepresence of functional FLP protein. Recombination takes place betweenidentical FRT sites such that the sequence between FRT sites in Oreplaces the sequence between the corresponding FRT sites of sequence G,thereby yielding a directionally targeted reintegrated new sequence. Thenew gene in O is now driven off of the P1 promoter in G. The purpose fordesigning some of the constructs without an ATG start codon on the geneis so that if random integration occurs, there is an extremely lowprobability of expression of the introduced gene, since in order forthis to happen, the fragment would need to integrate behind anendogenous promoter region and in the correct reading frame. This wouldoccur extremely rarely and our data to date have yielded no examples ofthis happening using a sequence such as O where the contained gene isthe easily scorable GUS gene. One requirement for each gene to beconstructed in this way (i.e., no ATG on the gene but with the ATGupstream of the FRT site) is the demonstration that the gene cantolerate a fusion of the FRT sequence between the ATG codon and thesecond codon of the protein. To date this has worked for quite a numberbut not all genes; in the latter cases the other form of the constructretaining the ATG (for example Q) could be used. All of the sequenceslisted above are expected to work in this scheme, some at differentfrequencies or efficiencies than others.

One problem this strategy addresses is limitations with currenttransformation approaches, particularly in plants, where delivery of DNAinto cells or nuclei and subsequent integration in the genome occursmore or less randomly and unpredictably. This is particularly true withparticle bombardment methods; arguments have been made thatAgrobacterium-based methods tend to deliver T-DNA border-flankedsequences to more actively transcribed regions of the genome, but beyondthat the process is still largely random. Therefore, for commercialproduct development, large numbers (estimates of >200) of events need tobe generated in order to identify one event: a) that expresses at thedesired level; b) where the gene product is functional and efficacious;c) which has a simple integration complexity to facilitate breeding; d)which does not contain extraneous sequences posing possible regulatoryconcerns; e) which maintains stability in expression over generations;f) most importantly, which does not have a negative impact on agronomicperformance characteristics when carried through a breeding programinvolving introgression of the trait into different genetic backgrounds.Resource utilization is very large and so schemes that can markedlyreduce the resource demand would be very beneficial to production oflarger numbers of desired final products.

Example 1 Creation of Novel Non-Identical FRT Sites

DNA fragments containing novel FRT sequences were constructed either bysynthesizing, annealing and ligating complementary oligonucleotides orby creating primers for PCR amplification (Mullis and Faloona, 1987) ofa DNA product containing the new FRT sequence near the 5′ end of the PCRproduct. The newly constructed FRT product includes flanking restrictionsites useful for cloning into plant expression units. In general, the 5′end is flanked by an NheI site and a terminal NcoI site. The NcoI siteincludes the bases ATG, which are advantageously used in newly developedvector constructs as the recognition sequence to initiate an openreading frame. In sequence-based constructs designated noATG/FRT, theNheI site is used for cloning thereby eliminating the upstream ATG inthe process. At the 3′ end of the FRT sequence, a restriction site isincluded enabling unique identification of the individual spacersequences. As specific examples, the wild type FRT site (designated FRT1here) is cloned with a flanking BglII site, the FRT5 site (spacerTTCAAAAG) (nt 39-46 of SEQ ID NO:3) has a ScaI site, the FRT6 site(spacer TTCAAAAA) (nt 36-49 of SEQ ID NO:4) has an AatII site, and theFRT7 site (spacer TTCAATAA) (nt 36-46 of SEQ ID NO:5) has an SpeI site.The outermost flanking restriction site is an XhoI site and is used toclone a gene of interest into the open reading frame.

The structures and sequences of the FRT sites as designed and/or used inthe present invention example are depicted below with positions ofrestriction sites, repeats and spacer regions indicated.

FRT1 (SEQ ID NO:2) Ncol      NheI   Repeat 1          Repeat2       Spacer    Inverted Repeat BglII XhoI 5′      CCATGGCTAGCGAAGTTCCTATTCC GAAGTTCCTATTC TCTAGAAA GTATAGGAACTTC AGATCTCGAG FRT5 (SEQID NO:3) Ncol      NheI   Repeat 1          Repeat2       Spacer    Inverted Repeat ScaI XhoI 5′      CCATGGCTAGCGAAGTTCCTATTCC GAAGTTCCTATTC TTCAAAAG GTATAGGAACTTC AGTACTCGAG FRT6 (SEQID NO:4) Ncol      NheI   Repeat 1          Repeat2       Spacer    Inverted Repeat AatII XhoI 5′      CCATGGCTAGCGAAGTTCCTATTCC GAAGTTCCTATTC TTCAAAAA GTATAGGAACTTC AGACGTCCTCGAG FRT7(SEQ ID NO:5) Ncol      NheI   Repeat 1          Repeat2       Spacer    Inverted Repeat SpeI XhoI 5′      CCATGGCTAGCGAAGTTCCTATTCC GAAGTTCCTATTCTTCAATAA GTATAGGAACTTCACTAGTTCTCGAG

Example 2 Creation of Plant Transformation Vectors Containing NovelNon-Identical FRT Sites

Based on the design of FRT sites as described above, PCR or standardmutagenesis protocols were used to create an XhoI site overlapping thestart of a gene sequence to be used for cloning downstream of the FRTsite, thereby converting the ATG start codon to GTG. Ligation of an FRTto the mutated gene sequence at XhoI creates a new open reading frameinitiating 5′ to the FRT. A second FRT sequence can be cloned downstreamof the terminator using a variety of methods including PCR or ligation.The FRT/gene/terminator/FRT unit can then be used to make target orsubstrate constructs.

Targets are created by inserting a promoter at the NcoI site upstream ofthe first FRT. This maintains a complete open reading frame of theFRT/gene fusion. These target constructs are for use in transformationexperiments to create desirable “target lines”. Substrate vectors areconstructed by cloning with the NheI site to truncate the start codon ofthe FRT/gene unit, thereby eliminating the proper open reading frame.These substrate vectors are used in experiments designed to retarget anew gene flanked by FRT sites into the corresponding FRT sitespreviously introduced in the target lines. In either case, to createmultiple gene cassettes, additional promoter/gene/terminator units areinserted between the terminator and the second FRT in either target orsubstrate molecules.

Example 3 Demonstration of Functionality of Novel FRT Sites andRequirement for Two Identical Sites for Efficient Recombination of DNASequences Positioned Between Two FRT Sites

Plasmids containing two identical or two different FRT sequences wereassayed for efficiency of recombination of sequences between the FRTsites by transformation into 294-FLP, a version of the E. coli strainMM294 with FLP recombinase integrated into the lacZ locus (Buchholz etal. 1996). Strains were grown overnight at 37° C. with shaking, allowingfor constitutive expression of FLP recombinase in the cultures. Theplasmid DNA was isolated using standard procedures and digested withrestriction enzymes that create novel restriction fragments followingFLP mediated recombination. The extent of recombination between FRTsites was estimated by examining banding patterns on an agarose gel.Table 1 summarizes data from the gel analysis.

TABLE 1 Target Site Combination Extent of Recombination FRT1 and FRT1Complete FRT5 and FRT5 Extensive, but partially incomplete FRT6 and FRT6Complete FRT7 and FRT7 Complete FRT1 and FRT5 No recombination FRT1 andFRT6 No recombination FRT1 and FRT7 No recombination FRT5 and FRT6 Norecombination FRT5 and FRT7 No recombination FRT6 and FRT7 Very smallamount of recombination

The results from these studies indicate that excision of sequencesbetween identical FRT sites occurs with high efficiency in general(FRT5, SEQ ID NO:3, appeared to be less efficient overall than FRT1, SEQID NO:2, or the novel FRT6, SEQ ID NO:4, and FRT7, SEQ ID NO:5, sites).As importantly, recombination with two different FRT sites was absent,or at least undetectable under the conditions of this assay for allcombinations but FRT6, SEQ ID NO:4, and FRT7, SEQ ID NO:5, where a smalldegree of recombination was noted. These data provided strong supportfor the potential utility of non-identical FRT sites in developing adirectional gene integration system. A point to note is that becauserecombination of sequences between two identical FRT sites can occurwith different efficiencies depending on the specific FRT site used(e.g., FRT5, SEQ ID NO:3, in the present experiment), the design ofconstructs for directional targeted integration may require judiciousselection of pairs of FRT sites to optimize for the desiredrecombination efficiency or to avoid any unwanted recombination.

Example 4 Introduction of DNA Sequences Which Include NovelNon-Identical FRT Sites into Plant Cells, Generation and Recovery ofStable Transgenic Events (“Target Lines”), Preservation of “TargetLines” and Regeneration of Plants

A number of stable transgenic events carrying FRT target sites wereproduced. These target lines were generated by introducing one of aseries of constructs including, for example, PHP9643, PHP10616,PHP11407, PHP11410, PHP11457, PHP11599, PHP11893 or PHP14220 (See Table2) into corn cells, either by particle bombardment, as described inRegister et al. (1994) Plant Mol. Biol. 25:951-961 or via Agrobacteriumco-cultivation as described by Heath et al. (1997) Mol. Plant-MicrobeInteract. 10:22-227; Hiei et al. (1994) Plant J. 6:271-282 and Ishida etal. (1996) Nat. Biotech. 14:745-750, and in U.S. Provisional ApplicationSer. No. 60/045,121 to “Agrobacterium Mediated Sorghum Transformation”,filed Apr. 30, 1997, now U.S. application Ser. No. 09/056,418, filedApr. 7, 1998. All vectors were constructed using standard molecularbiology techniques as described for example in Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual (2^(nd) ed., Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.). Table 2 below describes thecomponents within each of the vectors used to create a set of targetlines. The assembly strategy was as follows. The first expression unitin each case contains the 2.0 kb PstI fragment of the maize ubiquitinpromoter Ubi-1 (Christensen et al. (1992) Plant Mol. Biol. 18:675-689).Downstream of the ubiquitin promoter, varying FRT sequences wereinserted using NcoI or other sites that retained the ATG start codon.PHP10616 has the mo-PAT (U.S. Provisional Patent Application Ser. No.60/035,560 to “Methods for Improving Transformation Efficiency”, filedJan. 14, 1997 now U.S. Pat. No. 6,096,947) coding sequence fused inframe at the XhoI site flanking FRT1 (see above, SEQ ID NO:2). PHP11407and PHP11893 have GFPm-C3 (PCT/US97/07688 filed May 1, 1997 fromProvisional Application 60/016,345 filed May 1, 1996, now WO97/41228)containing the second intron from potato ST-LS1 (Vancanneyt et al.(1990) Mol. Gen. Genet. 220:245-250) fused in frame at the XhoI site ofFRT1 and FRT6, respectively. The potato proteinase inhibitor II (PinII)terminator (bases 2 to 310 from An et al. (1989) Plant Cell 1:115-122)was ligated downstream of the coding sequences. PHP10616 has an FRT5sequence (SEQ ID NO:3) cloned downstream of the PinII terminator.

TABLE 2 PHP Upstream-1 Coding-1 Downstream-1 Upstream-2 Coding-2Downstream-2 Coding-3 Downstream-3  9643 Ubiquitin ATG/FRT1E35S/35S/O′/ADH moPAT 35S term NoATG/FRT1/ pinII, FRT5 intron GFPm 10616Ubiquitin ATG/FRT1/moPAT pinII, FRT5 11407 Ubiquitin ATG/FRT1/GFPm-pinII Ubiquitin HM1 pinII, FRT5 C3-intron 11410 Ubiquitin ATG/FRT5E35S/35S/O′/ADH BAR 35S term, intron FRT1 11457 Ubiquitin ATG/FRT6E35S/35S/O′/ADH BAR 35S term, intron FRT1 11599 Ubiquitin ATG/FRT635S/O′/ADH intron BAR 35S term, FRT1 11893 Ubiquitin ATG/FRT6/GFPm-pinII Ubiquitin HM1 pinII, FRT1 C3-intron 14220 Ubiquitin/FRT1 FLPmpinII Ubiquitin GFPm pinII, FRT5 in 5′ UTR Ubiquitin/FRT1 FLPm pinIIUbiquitin GFPm pinII, FRT5 in intron

The second expression units have the maize ubiquitin promoter oralternatively either the enhanced or the standard versions of thecauliflower mosaic virus 35S promoter. The standard 35S promoterincludes bases −421 to +2 (from Gardner et al. (1981) Nucl. Acids Res.9:2871-2888), and the enhanced version has a duplication of bases −421to −90 upstream of this standard 35S promoter. The 79 bp tobacco mosaicvirus leader O′ (Gallie et al. (1987) Nucl. Acids Res. 15:3257-3273) isinserted downstream of the 35S promoter followed by the first intron ofthe maize alcohol dehydrogenase ADH1-S gene (Dennis et al. (1984) Nucl.Acids Res. 12:3983-3990). Coding sequences in these second expressionunits include either mo-PAT, bar (Thompson et al. (1987) EMBO J.6:2519-2523), or HM1 (Johal and Briggs, Science 258:985-987) genesfollowed by either the PinII terminator or the 35S terminator(nucleotides 7487-7639 in Gardner et al. (1981) Nucl. Acids Res.9:2871-2888). Varying FRT sites are ligated downstream of theterminators as shown in the table. A third expression unit is present inPHP9643 and has an FRT1/GFPm fusion cloned using the flanking NheI siteof FRT1 (SEQ ID NO:2) to remove the ATG start codon of GFPm, therebymaking it non-functional in the existing construct, but where correctexcision of sequences between FRT1 (SEQ ID NO:2) sites can bring theGFPm in frame with the ubiquitin promoter and ATG of the firstexpression unit, thereby making it functional. Downstream of GFPm is thePinII terminator followed by an FRT5 sequence (SEQ ID NO:3).

PHP9643 was cloned into a pUC derived plasmid backbone. All othervectors were cloned into a pSB11 (See, for example, EPA0672752A1,EPA0604662A1, EPA0687730A1 and U.S. Pat. No. 5,591,616) type plasmidwith the expression units contained between the TDNA border sequences.All are oriented with expression unit one adjacent to the right border.The pSB11-based plasmids were integrated into the super binary plasmidpSB1 (See, for example, EPA0672752A1, EPA0604662A1, EPA0687730A1 andU.S. Pat. No. 5,591,616) by homologous recombination between the twoplasmids. E. coli strain HB101 containing the pSB11 derivatives wasmated with Agrobacterium strain LBA4404 harboring pSB1 to create thecointegrate plasmids PHP10616, PHP11407, PHP11410, PHP11457, PHP11599,PHP11893 and PHP14220 in Agrobacterium (by the method of Ditta et al.(1980) Proc. Natl. Acad. Sci. USA 77:7347-7351). The cointegrates wereverified by Agrobacterium resistance to spectinomycin and SalIrestriction digests.

Table 2 also includes one example of a vector for creating a target linewhere the FRT sites are inserted in the maize ubiquitin intron (lastentry) as an alternative location for placement of FRT or other targetsites.

Following selection of stably transformed events, samples of thesetarget lines were cryopreserved as a supply for future experiments usingthe approach described by Peterson (see application Ser. No. 08/859,313,now U.S. Pat. No. 6,143,563). For several but not all events, anothersample callus from several of the stable transgenic events was grown,transferred onto regeneration medium to induce plantlet formation andplants were subsequently recovered and grown to maturity (Register etal. (1994) Plant Mol. Biol. 25:951-961).

Example 5 Demonstration of Functionality of Novel FRT Sites in Plants

(A) Excision of DNA Sequences Between Two Identical FRT Sites, But NotWhen Flanked by Two Non-Identical FRT Sequences

The extent of intra-plasmid recombination was examined in plants usingthe FRT excision constructs described in Table 3 below. The vectorsPHP10968, PHP10998, PHP10969, PHP11272, PHP11243, PHP11244, PHP12140,PHP12141, PHP12156, and PHP12157 were constructed by ligating the maizeUbiquitin promoter upstream of FRT sequences using NcoI or other sitesthat maintained the ATG start codon. The FRT sequence was fused in frameat the flanking XhoI site to a GFPm sequence containing a serine tothreonine mutation at amino acid residue 65 in the wild type sequence(new sequence termed GFPm-S65T). The pinII terminator was cloneddownstream of GFPm. The second expression unit consists of apromoterless FRT, cloned with the 5′ flanking NheI site to remove theATG start codon, fused in frame to the GUS coding sequence (Jefferson etal. (1986) Proc. Natl. Acad. Sci. USA 83: 8447-8451) and followed by thepinII terminator. The vector backbone is a pUC derived plasmid in allcases. Experiments were conducted by bombarding the indicated plasmidsinto maize cells along with construct PHP5096, which carries afunctional expression cassette for FLP protein. PHP5096, the FLPmexpression vector that was used in experiments with the excision andsubstrate vectors, consists of the maize Ubiquitin promoter clonedupstream of the FLPm coding sequence (U.S. patent application Ser. No.08/972,258 to “Novel Nucleic Acid Sequence Encoding FLP Recombinase” nowU.S. Pat. No. 5,929,301) and the pinII terminator in a pUC derivedplasmid backbone. In each case, successful excision would removeintervening sequences between the indicated FRT sites thereby bringingan inactive uidA (GUS) gene in frame with and in proximity to theubiquitin promoter resulting in GUS activity. If excision does notoccur, no GUS expression is expected. The results for GUS expressionfrom these experiments are indicated in Table 4 below. In these studiesefficient excision occurred only where constructs contained twoidentical FRT sites. In the case of the FRT6 (SEQ ID NO:4) and FRT7 (SEQID NO:5) combination, a small amount of recombination was observed,again emphasizing the need for testing target site combinations andjudiciously selecting appropriate combinations for the application.

TABLE 3 PHP Upstream-1 Coding-1 Downstream-1 Upstream-2 Coding-2Downstream-2 10968 Ubiquitin ATG/FRT1/GFPm-S65T PinII noATG/FRT1/GUSpinII 10998 Ubiquitin ATG/FRT5/GFPm-S65T PinII noATG/FRT5/GUS pinII11272 Ubiquitin ATG/FRT6/GFPm-S65T PinII noATG/FRT6/GUS pinII 12157Ubiquitin ATG/FRT7/GFPm-S65T PinII noATG/FRT7/GUS pinII 10969 UbiquitinATG/FRT1/GFPm-S65T PinII noATG/FRT5/GUS pinII 11243 UbiquitinATG/FRT1/GFPm-S65T PinII noATG/FRT6/GUS pinII 12140 UbiquitinATG/FRT1/GFPm-S65T PinII noATG/FRT7/GUS pinII 11244 UbiquitinATG/FRT5/GFPm-S65T PinII noATG/FRT6/GUS pinII 12141 UbiquitinATG/FRT5/GFPm-S65T PinII noATG/FRT7/GUS pinII 12156 UbiquitinATG/FRT6/GFPm-S65T PinII noATG/FRT7/GUS pinII 12933 Ubiquitin/FRT1 in 5′UTR GFPm-S65T PinII FRT1 in 5′ UTR/Ubi intron GUS pinII 14076Ubiquitin/FRT1 in intron AHAS PinII FRT1 in Ubi intron GUS pinII 14053Ubiquitin/FRT1 in intron AHAS PinII FRT5 in Ubi intron GUS pinII 14086Ubiquitin/FRT1 in intron AHAS PinII FRT6 in Ubi intron GUS pinII

TABLE 4 Recombination tested GUS Plasmid between expression PHP10968FRT1 and FRT1 +++ PHP10998 FRT5 and FRT5 ++ PHP11272 FRT6 and FRT6 +++PHP12157 FRT7 and FRT7 +++ PHP9643 FRT1 and FRT5 − PHP11243 FRT1 andFRT6 − PHP12140 FRT1 and FRT7 − PHP11244 FRT5 and FRT6 − PHP12141 FRT5and FRT7 − PHP12156 FRT6 and FRT7 +B) Transient Integration of a Second DNA Sequence Flanked by TwoNon-Identical FRT Sequences into Plant Cells

Summarized in Table 5 below are data from experiments in which targetlines created using the plasmids described in Table 2 were bombardedwith a substrate plasmid containing a GUS reporter gene flanked by thecorresponding FRT sites used in the target constructs. This experimentmeasured the ability to detect transient GUS expression shortly afterintroduction of the substrate plasmid. Since there is no promoter infront of the first coding sequence in the substrate plasmids, randomintegration, unless occurring in frame behind an appropriate regulatorysequence elsewhere in the genome, would not result in GUS expression.This assay system then evaluates the ability to target FRT-flanked genesinto FRT sites in the genome. In general, FRT substrate vectors (Table6) are constructed as promoterless FRT/gene fusions cloned using the 5′flanking NheI site of the FRT to remove the ATG start codon. Genes fusedin frame to the FRT with the flanking XhoI site include one of severalscorable or selectable marker genes such as aadA (Svab et al. (1990)Plant Mol. Biol. 14: 197-205), uidA, GFPm, GFPm-C3/intron or bar and arefollowed by a pinII terminator. In some cases (PHP10259, PHP10603,PHP11561, and PHP11633), plasmids contain a single expression unit andthe second heterologous FRT site is cloned downstream of the pinIIterminator. Substrate plasmids PHP10859, PHP10997, PHP11204, PHP11699,and PHP12190 have in addition to the first expression unit describedabove, a second unit consisting of the maize ubiquitin promoter, theenhanced 35S promoter or a chimeric promoter consisting of the 35Senhancer region cloned upstream of a synthetic core promoter termedRsyn7 (U.S. Pat. No. 6,072,050 which is a continuation in part of U.S.patent application Ser. No. 08/661,601 filed Jun. 11, 1996 nowabandoned) cloned upstream of either the HM1, aadA, GUS, or bar codingsequences and the pinII terminator. A heterologous FRT is inserteddownstream of the second terminator. Finally, PHP11003 and PHP11809contain three expression units. The first unit is a promoterlessnoATG/FRT/gene fusion as described above, the second unit containseither the chimeric 35S enhancer/Rsyn7 promoter described above or theZmdJ1 promoter (Baszczynski et al. (1997) Maydica 42:189-201) clonedupstream of the GUS coding sequence and the pinII terminator. The thirdexpression unit consists of the maize ubiquitin promoter cloned upstreamof the HM1 coding sequence, pinII terminator and a heterologous FRTsequence. All FRT substrate vectors are cloned into a pUC derivedplasmid backbone. Details of the components of these vectors aredescribed in Table 6. Also listed in Table 6 are two vectors withalternative placement of FRT sites in the ubiquitin 5′ UTR or intron.

TABLE 5 # of GUS PHP9643 PHP11147 PHP11410 PHP11407 PHP11457 Spots (n =74) (n = 127) (n = 32) (n = 38) (n = 113) no spots 17.57% 3.15% 6.25%2.63% 7.96%  1–25 22.97% 48.03% 62.50% 10.53% 27.43%  26–100 31.08%37.80% 18.75% 18.42% 32.74% 101–200 14.86% 8.66% 12.50% 57.89% 27.43%too many to 13.51% 2.36% 0.00% 10.53% 4.42% count

TABLE 6 PHP Coding-1 Downstream-1 Upstream-2 Coding-2 Downstream-2Upstream-3 Coding-3 Downstream-3 10259 NoATG/FRT1/aadA pinII, FRT5 10603NoATG/FRT1/GUS pinII, FRT5 10859 NoATG/FRT1/GFPm PinII Ubiquitin HM1pinII, FRT5 10997 NoATG/FRT5/GUS PinII Ubiquitin aadA pinII, FRT5 11003NoATG/FRT1/GFPm PinII E35S/Rsyn7/O′/ GUS pinII Ubiquitin HM1 pinII, FRT5ADH intron 11204 NoATG/FRT1/BAR PinII E35S/Rsyn7/O′/ GUS pinII, FRT5 ADHintron 11561 NoATG/FRT6/GUS pinII, FRT1 11633 NoATG/FRT5/GUS pinII, FRT111699 NoATG/FRT6/GFPm- PinII Ubiquitin HM1 pinII, FRT1 C3-intron 11809NoATG/FRT6/GFPm- PinII F3.7 GUS pinII Ubiquitin HM1 pinII, FRT1C3-intron 12190 NoATG/FRT1/GUS PinII E35S/35S/O′/ADH BAR pinII, FRT5intron Ubiquitin/FRT1 in HM1 pinII E35S/35S/O′/ADH BAR pinII, FRT5 5′UTR intron Ubiqutin/FRT1 in HM1 pinII E35S/35S/O′/ADH BAR pinII, FRT5intron intronResults in Table 5 indicate that the frequency and level of GUSexpression varies among different events, as might be predicted forgenes inserted in different positions in the genome. The prediction isthat once a high frequency, high expressing line is identified, that theexpression of genes subsequently introduced into those same sites willalso be higher than in other lower expressing events.C) Stable Integration of a Second DNA Sequence Flanked by TwoNon-Identical FRT Sequences into Plant Cells

A subset of the stable transgenic “target lines” described in example 4above was used in experiments aimed at stably retargeting into theseprimary target lines a new gene flanked by the same FRT sites used inthe target lines and cloned in a second construct “substrate” plasmid.Table 7 lists the constructs contained in the primary target lines (fromTable 2), the FRT sites contained in these lines and the substrateplasmids (from Table 6) that were subsequently retargeted into thetarget lines.

Table 8 presents data from stable transgenic events which demonstratesuccessful and reproducible targeting of introduced sequences topreviously created genomic target sites. The data shown are for 18independent target lines, each retargeted with a promoterless GUSconstruct. Since the bar gene was concurrently introduced on the sameplasmid, the proportion of GUS expressing events from the total eventsrecovered on bialophos selection provides a measure of retargetingfrequency relative to random integration.

TABLE 7 Target construct FRT sites Substrates being evaluated PHP96431/1/5 10603, 10259, 10859, 10997, 11003 PHP11147 1/5 10603, 10859, 11003PHP11407 1/5 10603, 11204, 12190 PHP11410 5/1 11633 PHP11457 6/1 11561,11699, 11809 PHP11893 6/1 Experiments in progress

TABLE 8 Target # of Random # of Targeted Targeting Line Events EventsFrequency (%) A 13 1 7.1 B 14 1 6.7 C 108 14 11.5 D 18 1 5.3 E 14 2 12.5F 9 1 10.0 G 65 1 1.5 H 63 9 12.5 I 71 6 7.8 J 15 1 6.3 K 33 9 21.4 L 192 9.5 M 8 1 11.1 N 12 1 7.7 O 29 4 12.1 P 43 4 8.5 Q 16 3 15.8 R 4 120.0 S 12 1 7.7 T 10 1 9.1 U 1 2 66.7

Example 6 Evaluation of Impact of Introduced FRT Sequences on PlantDevelopment, Gene Expression and Agronomic Performance

Initial evaluation of the impact of the introduced sequences on plantgrowth and gene expression is conducted in the greenhouse by makingregular observations through to pollination and seed set. Plants areboth selfed and crossed to other genotypes to obtain T1 seed forsubsequent greenhouse and field evaluation. For gene expressionevaluation, both qualitative and quantitative data are collected andanalyzed. T1 seeds from transgenic events which give acceptable ordesirable levels of expression and which show no significant negativeimpact on plant development (e.g., have normal developmental morphology,are male and female fertile, etc.) are then grown in managed field plotsalong with non-transgenic control plants, and standard agronomicperformance data is collected and evaluated.

Example 7 Conversion of an Introduced Functional FRT Sequence into aSecond Non-Identical Functional FRT Sequence

The approach taken here to develop a method for converting betweendifferent FRT sites for use in various applications is based on thepreviously described “chimeraplasty” strategy for making specifictargeted nucleotide modifications at a specified extrachromosomal orgenomic target sequence in animal cells (Yoon et al. (1996) Proc. Natl.Acad. Sci. 93:2071-2076; Cole-Strauss et al. (1996) Science273:1386-1389). This capability in plants, as demonstrated recently inour laboratories and described in WO99/25853, published May 27, 1999, isbeneficial to extending the potential use of the present invention forbroader application. The proposed use of this “chimeraplasty” technologyin the present invention would be to target and modify nucleotides inone FRT site of a pair of non-identical FRT sites flanking a DNAsequence of interest in a way that then makes the two FRT sitesidentical. Subsequent or concurrent expression of FLP recombinase incells with these FRT site modifications would lead to excision of thesequences between these now identical FRT sites, thereby removingspecifically the undesirable DNA sequences from the previously createdstable transgenic event containing those sequences. An application ofthis approach would be for example in the case of a selectable markerwhich is required during initial steps of a breeding or backcrossingprogram to maintain and select for preferred individual plants, butwhich is not desired in the final product.

A) Vector Design and Construction for Testing Chimeraplasty-Based FRTSite Conversion

The target vectors for evaluating this FRT site modification strategyare shown generically below, where P1 and P2 represent two differentpromoters, G1 and G2 represent two genes, and T1 and T2 represent twoterminator regions; these regions are shown as white boxes. DifferentFRT sites are indicated and shown as dark boxes. One version of theconstruct incorporates a third unique FRT site downstream of the secondgene and is used to evaluate whether the targeted conversion, in thiscase, of FRT5 to FRT6 (SEQ ID NO:4), also results in conversion of thedownstream FRT1 (SEQ ID NO:2) site to an FRT6 (SEQ ID NO:4) site. In theformer case, expression of the downstream gene (G1) should be detected,while if the conversion is not specific to FRT5 (SEQ ID NO:3) and theFRT1 (SEQ ID NO:2) site is converted also, then both gene activitieswill be lost. For the specific examples used here P1 is the maizeubiquitin promoter, P2 is the enhanced CaMV 35S promoter, G1 is the uidA(GUS) gene, G2 is the bar gene, and T1 and T2 are pinII terminators. Itis understood that based on the various descriptions of vectorconstructs earlier in this application, a variety of differentpromoters, genes, terminators or DNA sequences or FRT sites could beused in practicing this component method. The DNA cassettes as shownbelow could be assembled into either a pUC-based plasmid for direct DNAdelivery methods (such as particle bombardment) or into a binary vectorfor Agrobacterium-based transformation as described previously.

B) Design of Chimeric Oligonucleotide Molecules for Chimeraplasty-BasedTargeted Conversion of an FRT Site

Shown below are specific examples of chimeric molecules that would beused to modify a single nucleotide so as to convert the FRT5 (SEQ IDNO:3) site to an FRT6 (SEQ ID NO:4) site in constructs as describedabove. Both the linear sequence of these chimeric molecules as well asthe predicted active form of the molecule (based on the Yoon et al. andCole-Strauss et al. publications above) are shown. DNA residues arerepresented in upper case, RNA residues in lower case, and the site tobe modified (a single nucleotide difference between FRT5, SEQ ID NO:3,and FRT6, SEQ ID NO:4) is underlined and in bold. Two examples ofchimeras are presented below differing in the number of residuesdownstream of the FRT5 (SEQ ID NO:4) site that would be included in thechimeric molecule design and which would thus determine the specificityto the target sequence.

1. Chimeric oligonucleotide linear sequence (sequence includes sixtarget-specific residues downstream of the FRT site being modified inthe target construct and should convert only this single specific FRT5,SEQ ID NO:3, site to an FRT6, SEQ ID NO:4, site)

(SEQ ID NO:6) CCTATTCTTCAAAA A GTATAGGAACTTCAGTACTTTTTaguacugaaguuCCTATACTTTuugaagaauaggGCGCGTTTTCGCGC-3′

Active oligonucleotide conformation of SEQ ID NO:6

 TGCGCG- -ggauaagaaguuTTTCATATCCuugaagucaugaTT                                            TT                                            T  TCGCGC CCTATTCTTCAAAA AGTATAGGAACTTCAGTACTT          3′ 5′

2. Chimeric oligonucleotide linear sequence (sequence contains residuesspecific to only sequences in the FRT site and so should convert anyFRT5, SEQ ID NO:3, site in a target molecule to an FRT6, SEQ ID NO:4,site)

(SEQ ID NO:7) 5′- TATTCTTCAAAAAGTATAGGAACTTCTTTTgaaguuccuaTACTTTuugaagaauaGCGCGTTTTCGCGC-3′

Active oligonucleotide conformation of SEQ ID NO:7

 TGCGCG--auaagaaguuTTTCATauccuugaagTT                                      TT                                      T  TCGCGCTATTCTTCAAAAAGTATAGGAACTTCT      3′ 5′Vector constructions and chimeric oligonucleotide molecules as describedabove were generated and used in experiments.C) Demonstration of Conversion from One FRT Site to Another

Stable transgenic maize lines are generated with the constructs asdescribed above or with other related ones by transforming in theconstructs and selecting on bialophos as described before. Tissues to beused for chimera delivery are transferred onto non-bialophos-containingmedia and the chimeric oligonucleotides are delivered into cells ofthese stable events by particle bombardment, together with co-deliveryof PHP5096 which carries a functional FLP recombinase expressioncassette. In control experiments, only chimeric molecules or onlyPHP5096 are delivered. After sufficient time for cells to recoverwithout bialophos selection, samples of the bombarded events areevaluated for GUS expression. For those bombarded events containing theconstruct with the downstream FRT1 (SEQ ID NO:2) site which do not showGUS expression, an equivalent sample of cells are plated and grown onmedium with or without bialophos selection to assess sensitivity to thechemical. If the chimeric molecules are specific for modifying only theFRT5 (SEQ ID NO:3) site, then no differences in number and growth ofcells should be observed between treatments with or without selection.Otherwise, reduced growth and recovery should be noted.

D) Molecular Verification of Stable Conversion of FRT Sites

DNA from those samples that exhibit GUS expression is isolated,amplified by PCR if necessary, and sequenced by standard methods throughthe region corresponding to the predicted nucleotide conversion. Asufficient stretch of DNA is sequenced to cover the entire originallyintroduced region of DNA so as to confirm correct and specificconversion. Using standard methods for PCR, Southern analysis and/orsequencing of GUS expressing and non-expressing samples establishes thepresence or absence of specific DNA fragments prior to and followingchimeric molecule and FLP recombinase delivery, and thus substantiatesthe visual and biochemical observations made above.

E) Utility of Chimeraplasty-Based FRT Site Conversion in a TransgeneStacking Strategy for Plants

Described in FIG. 1 is one potential strategy for combining or stackingmultiple desired transgenes at one genomic location using thenon-identical FRT-based system of the present invention. While stackingof genes can be achieved without the use of the targeted FRT conversionmethod described in this example 7, this latter method extends thecapabilities of the system by allowing in vivo conversion of FRT sitesto create new sites, rather than re-introducing new FRT sites bytransformation. In the diagram of FIG. 1, an FRT site with an asteriskbeside it indicates that it was initially created to be non-functionalwith respect to recombination between it and the equivalent FRT sitewithout an asterisk, but which upon conversion with thechimeraplasty-based approach described herein renders it capable ofrecombination with its equivalent non-asterisk counterpart. In thespecific example presented in the figure, this would facilitate forexample removal of a selectable marker either to no longer have itpresent, or to allow one to re-use the selectable marker in futuretransformations. Thus this method also provides a mechanism to recycleselectable markers, as is possible in using the FRT system of thepresent invention alone.

Discussion

To date in plants, the major application of the FLP/FRT system has beenfor DNA excision (Lyznik et al. (1993) Nucleic Acids Res. 21:969-975).For example, a gene such as a selectable marker flanked by FRT sites isfirst introduced into plant cells by one of several transformationapproaches, and stable transgenic events or plants are recovered viaappropriate selection. Then in order to eliminate the selectable markergene, FLP protein is expressed in the cells either transiently byintroducing a plasmid carrying a FLP expression cassette, stablyfollowing integration of an introduced FLP expression cassette, or bycrossing plants carrying the FRT-flanked selectable marker gene withplants carrying sequences for and expressing active FLP protein(WO99/25841, published May 27, 1999, to “Novel Nucleic Acid SequenceEncoding FLP Recombinase”).

A major problem associated with developing the FLP/FRT system forintegrating genes into animals or plants stems from the fact that therecombination reaction catalyzed by yeast FLP recombinase is areversible process (Sadowski et al. (1995) in Progress in Nucleic AcidResearch and Molecular Biology 51:53-91). For example, followingintroduction of a DNA sequence flanked by similarly oriented FRT sitesinto plant cells in the presence of actively expressing FLP recombinase,recombination should lead to insertion of the new DNA sequences at theendogenous FRT site. However, with continued expression of FLP enzyme,the reverse reaction would lead to re-excision of the introducedsequences because of recombination between the identical FRT sites.Since the reaction is reversible, integration and excision canrepeatedly continue towards equilibrium. As cells divide and the DNAsubstrate concentration per cell decreases, the probability ofintegration decreases, such that in general, as long as active FLPprotein is expressed the reaction will be driven towards thenon-integrated state. To favor integration, a situation must beestablished which precludes re-excision once integration occurs. Anumber of strategies have been suggested, including limiting theduration of activity of FLP recombinase through inducible expression orby directly introducing FLP protein or RNA into cells (Sadowski et al.(1995) Progress on Nucleic Acid Research and Molecular Biology51:53-91), but to date no routine non-random integration system has beenestablished for plants.

The present invention describes the development of a useful new genetargeting system for plants which utilizes the yeast FLP recombinase ora modified FLP recombinase designed to work more efficiently in certainplant species and novel non-identical FRT sites which can be used fordirectional non-reversible DNA integration. Additionally, describedherein is a novel use of accessory technologies such as “chimeraplasty”permitting in vivo or in vitro modification of DNA sequences, such asFRT sites to further extend the utility of the system. Data provideddemonstrate the successful stable integration of DNA sequences betweentwo previously introduced non-identical FRT sites in maize. We show alsothat the DNA sequences between the FRT sites can be subsequentlyreplaced by a second DNA sequence flanked by the same FRT sites as thefirst. Together these results demonstrate that it is possible tointroduce and recover pairs of non-identical FRT sites at certaingenomic locations, that one can select desirable or preferred genomiclocations for expressing DNA sequences of interest, and that theseselected locations can be used to re-target other DNA sequences ofinterest. Apart from the obvious benefits of being able to integrategenes into the genome of plants, the present invention provides a meansfor facilitating the introduction of novel genes or DNA sequences intogenomic locations previously determined to be particularly beneficialfor gene integration from the perspective of providing suitable levelsof stable expression of the introduced gene(s) and not exhibitingdeleterious impacts on agronomic characteristics including yield. Inaddition the invention provides a system whereby integration of two ormore genes can be targeted to the same genomic location, providing amechanism for “gene stacking”. These stacked genes can then bemaintained and managed as a closely linked pair of traits in breedingprograms. Thus this invention also provides an improved method forintroducing, maintaining and breeding multiple genetic traits ofinterest, including agronomic traits, commercially important genes orother heterologous gene products.

The invention further proposes to use the non-recombination feature ofnon-identical FRT sites to allow creation of a set of ‘parental’ lines,which are initially well-characterized for all the desired expressionand performance parameters described above. These lines then serve asthe basis for introduction of new traits into the same predefined sitesin the genome where the initial genes were introduced. Many fewer eventswould need to be generated, since integration would preferentially occurin sites shown to express well and have minimal negative impact onperformance.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A plant cell having stably incorporated intoits genome at least a first and a second transfer cassette, wherein saidfirst transfer cassette comprises a first nucleotide sequence ofinterest flanked by a first and a second recombination site, whereinsaid first and said second recombination sites are non-identical; saidsecond transfer cassette comprises a second nucleotide sequence ofinterest flanked by the second recombination site and a thirdrecombination site, wherein said third recombination site isnon-identical to said first and said second recombination site; and,said second recombination site is shared between the first and thesecond transfer cassette, such that the genome of the plant cellcomprises in the following order the first recombination site, the firstnucleotide sequence of interest, the second recombination site, thesecond nucleotide sequence of interest, and the third recombinationsite, wherein said first, said second and said third non-identicalrecombination sites can recombine with their identical recombinationsites in said plant cell in the presence of an appropriate recombinase.2. The plant cell of claim 1, wherein said first, said second, or saidthird recombination site is selected from the group consisting of a FRTsite, a mutant FRT site, a LOX site and a mutant LOX site.
 3. The plantcell of claim 2, wherein the first, the second, or the thirdrecombination site is selected from the group consisting of a FRT siteand a mutant FRT site.
 4. The plant cell of claim 3, wherein the mutantFRT site is selected from the group consisting of FRT 5 (SEQ ID NO:3),FRT 6 (SEQ ID NO:4), and FRT 7 (SEQ ID NO:5).
 5. The plant cell of claim1, wherein said plant cell is within a plant.
 6. The plant cell of claim1, wherein said plant cell is within a seed.
 7. The plant cell of claim1, wherein said plant cell is from a monocot.
 8. The plant cell of claim7, wherein said monocot is maize.
 9. The plant cell of claim 1, whereinsaid plant cell is from a dicot.
 10. The plant cell of claim 9, whereinsaid dicot is canola, Brassica, soybean, sunflower, or cotton.
 11. Theplant cell of claim 1, wherein said first nucleotide sequence ofinterest is operably linked to an inducible promoter.
 12. The plant cellof claim 1, wherein said first and said second and said thirdnon-identical recombination site can recombine with their identical sitein the presence of the same recombinase.
 13. The plant cell of claim 1,wherein said first, said second and said third non-identicalrecombination sites can recombine with their identical recombinationsites when their identical recombination sites are sexually crossed intosaid plant cell.
 14. The plant cell of claim 1, wherein said plant cellhas stably incorporated into its genome a polynucleotide comprising inthe following order, at least one expression cassette comprising a thirdnucleotide sequence of interest, the first transfer, and the secondtransfer cassette.